Loss of MAFB Function in Humans and Mice Causes Duane Syndrome, Aberrant Extraocular Muscle Innervation, and Inner-Ear Defects  Jong G. Park, Max A. Tischfield, Alicia A. Nugent, Long Cheng, Silvio Alessandro Di Gioia, Wai-Man Chan, Gail Maconachie, Thomas M. Bosley, C. Gail Summers, David G. Hunter, Caroline D. Robson, Irene Gottlob, Elizabeth C. Engle  The American Journal of Human Genetics  Volume 98, Issue 6, Pages 1220-1227 (June 2016) DOI: 10.1016/j.ajhg.2016.03.023 Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 1 Mutations in MAFB Cause DRS (A) MAFB mutations segregate with DRS in pedigrees FA, PM, 0819, and N. Three of the four affected FA pedigree members also have unilateral or bilateral congenital hearing loss (indicated by an asterisk). (B) FA IV:1 has bilateral DRS, characterized by bilaterally limited eye abduction and narrowing of the palpebral fissures with globe retraction during attempted eye adduction. (C) Axial CT images of the right temporal bone of a healthy control individual with normal cochlea and vestibule (arrows) and of individual FA IV:1, who has a cystic common-cavity anomaly (arrow). The American Journal of Human Genetics 2016 98, 1220-1227DOI: (10.1016/j.ajhg.2016.03.023) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 2 Mafb-Mutant Mouse Embryos Demonstrate DRS Pathology (A–F) Whole-mount sagittal confocal images at E11.5. (A) MafbWT/WT embryos showed normal hindbrain and cranial nerve development. The white line indicates the region of developing rhombomeres 5 and 6. (B) MafbWT/KO embryos had abducens nerve hypoplasia but no other major abnormalities of hindbrain and cranial nerve development. (C) MafbKO/KO embryos showed loss of rhombomeres 5 and 6, resulting in loss of the hindbrain area (white line), an absent abducens nerve, and fusion of the glossopharyngeal nerve and the vagus nerve (arrow). (D–F) Medial sagittal sections highlight the developing oculomotor and abducens nerves. (D) In MafbWT/WT embryos, the abducens nerve was present (short arrow) and reached the developing eye. (E) MafbWT/KO embryos had a hypoplastic abducens nerve (short arrow). (F) MafbKO/KO embryos were missing an abducens nerve (short arrow). (G–O) Confocal images of the right orbit in mouse embryos from the inferior view. (G) At E12.5 in MafbWT/WT embryos, the abducens nerve (arrowhead) innervated the LR muscle, whereas the oculomotor nerve innervated the IR muscle and developing IO muscle. (H) In E12.5 MafbWT/KO embryos, the abducens nerve was hypoplastic (arrowhead) and innervated the LR muscle, whereas the oculomotor nerve began to send aberrant branches toward the LR muscle (arrow) and the RB muscle. (I) In E12.5 MafbKO/KO embryos, the abducens nerve was absent (arrowhead), and the oculomotor nerve sent aberrant branches toward the LR muscle (arrow) and the RB muscle. (J) At E13.5 in MafbWT/WT embryos, the abducens nerve (arrowhead), oculomotor nerve, and extraocular muscles continued to develop normally. (K) In E13.5 MafbWT/KO embryos, the hypoplastic abducens nerve (arrowhead) innervated the LR muscle, whereas the oculomotor nerve formed a second, more distal aberrant branch toward the LR muscle (yellow arrow), in addition to the proximal aberrant branch formed earlier (white arrow). (L) In E13.5 MafbKO/KO embryos, the abducens nerve was absent (arrowhead), and the oculomotor nerve sent a distinct distal aberrant branch toward the LR muscle (yellow arrow), in addition to the proximal aberrant branch formed earlier (white arrow) and the other aberrant branches contacting the RB muscle. (M) MafbWT/WT embryos at E16.5 had the final developmental pattern of the orbit, whereby the abducens nerve (arrowhead) innervated the LR muscle. (N) In E16.5 MafbWT/KO embryos, the abducens nerve remained hypoplastic (arrowhead), and a proximal aberrant branch (white arrow) and a distal aberrant branch (yellow arrow) of the oculomotor nerve also innervated the LR muscle. (O) In E16.5 MafbKO/KO embryos, the abducens nerve remained absent (arrowhead), and a proximal aberrant branch (white arrow) and a distal aberrant branch (yellow arrow) of the oculomotor nerve innervated the LR muscle instead. (P) At E16.5, the diameter of the abducens nerve was significantly smaller in MafbWT/KO embryos than in MafbWT/WT embryos and was absent in MafbKO/KO embryos. (Q) At E16.5, the diameter of the oculomotor distal aberrant branch was significantly greater than that of the oculomotor proximal aberrant branch in both MafbWT/KO and MafbKO/KO embryos. The diameter of the distal aberrant branch was significantly greater in MafbKO/KO than in MafbWT/KO embryos. These aberrant branches were not present in MafbWT/WT embryos. Abbreviations are as follows: III, oculomotor nerve; IV, trochlear nerve; V, trigeminal nerve; VI, abducens nerve; VII, facial nerve; IX, glossopharyngeal nerve; X, vagus nerve; IO, inferior oblique; IR, inferior rectus; LR, lateral rectus; and RB, retractor bulbi. Scale bars represent 100 μm. ∗p < 0.05, ∗∗∗∗p < 0.0001; differences were measured by Tukey’s multiple-comparison test, and error bars represent the SEM (GraphPad Prism). n > 10 for each genotype. The American Journal of Human Genetics 2016 98, 1220-1227DOI: (10.1016/j.ajhg.2016.03.023) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 3 Less Than 50% MAFB Function Causes DRS and Inner-Ear Defects (A) Pedigree N has a full gene deletion and therefore no mutant MAFB. Pedigrees 0819 and PM are predicted to have truncated MAFB proteins that lack the EHR, BR, and LZ domains followed by 78 and 10 altered amino acids, respectively. Pedigree FA is predicted to have a MAFB that retains the wild-type EHR, BR, and beginning of the LZ followed by 125 altered amino acids. (B) A luciferase assay showed that wild-type MAFB increased transcription by approximately 150-fold. The 0819 or FA mutant protein alone did not have any transcriptional activity. Co-expression of wild-type MAFB and the FA mutant, but not the 0819 mutant, reduced the transcriptional activity of the wild-type protein in comparison to that of the wild-type alone. ∗∗p < 0.01; differences were measured by Tukey’s multiple-comparison test, and error bars represent the SEM (GraphPad Prism). Each experiment was performed in triplicate. (C) Threshold model for loss of MAFB function. At greater than 50% MAFB function, MafbWT/WT and kr/+ mice had no phenotypic alterations. At 50% MAFB function, MafbWT/KO mice and N, 0819, and PM family members with heterozygous loss-of-function mutations had isolated DRS. At some level below 50% MAFB function, kr/kr mice, FA members with a dominant-negative mutation, and MafbKO/KO mice had both DRS and inner-ear defects. The American Journal of Human Genetics 2016 98, 1220-1227DOI: (10.1016/j.ajhg.2016.03.023) Copyright © 2016 American Society of Human Genetics Terms and Conditions